Our first aim is to elucidate the mechanism of action of Hsp90 and the interplay between Hsp90 and the Hsp70 chaperone system. The Hsp90 family of heat shock proteins represents one of the most abundantly expressed and highly conserved families of molecular chaperones. Eukaryotic Hsp90 is known to control the stability and the activity of several hundred client proteins. Moreover, it is important for the growth and survival of cancer cells, and drugs targeting Hsp90 are currently in clinical trials. To gain insight into the mechanism of action of this important family of chaperones, we are studying Hsp90 from E. coli (Hsp90Ec) and yeast (Hsp82). We discovered that Hsp90Ec and the E. coli Hsp70 chaperone system (DnaK, DnaJ and GrpE) act synergistically in protein reactivation in vitro and that Hsp90Ec and DnaK directly interact in the absence of cochaperones. Additionally, we have demonstrated that a region of Hsp90Ec in the middle domain of the protein is important for the interaction with DnaK. By using molecular docking, we identified a region in the nucleotide-binding domain of DnaK, when DnaK was in the ADP bound conformation, that was predicted to interact with the middle domain of Hsp90Ec. We made substitution mutants in DnaK residues predicted from the model to interact with Hsp90Ec and found that most of the mutants were defective or partially defective in their ability to interact with Hsp90Ec in vivo and in vitro. The region of DnaK we identified as important for the interaction with Hsp90Ec overlaps with the region of DnaK that interacts with the J-domain of DnaJ. We also found that yeast Hsp82 and Hsp70 (Ssa1) directly interact in vitro in the absence of the yeast Hop homolog, Sti1, which was thought to be a bridging protein between Hsp82 and Ssa1 prior to this work. We identified a region in the middle domain of yeast Hsp90 that is required for the interaction with Ssa1 by constructing and analyzing Hsp82 mutants in residues homologous to those we had identified in Hsp90Ec as being important for interaction with DnaK. In vivo results using Hsp82 substitution mutants showed that several residues in this region were important or essential for growth at high temperature. Moreover, mutants in this region were defective in interaction with Ssa1 in cell lysates. In vitro, the purified Hsp82 mutant proteins were defective in direct physical interaction with Ssa1 and in protein remodeling in collaboration with Ssa1 and cochaperones. This region of Hsp90 is also important for interactions with several Hsp90 cochaperones and client proteins, suggesting that collaboration between Hsp70 and Hsp90 in protein remodeling may be modulated through competition between Hsp70 and Hsp90 cochaperones for the interaction surface. However, the results showing that Hsp90 and Hsp70 directly interact did not distinguish between the possibility that (i) these sites were involved in direct interaction and (ii) the residues in these sites participated in conformational changes which were transduced to other sites on Hsp90 and Hsp70 that were involved in the direct interaction. Thus we performed crosslinking experiments. The results showed that the direct interaction between the two chaperones is between a site in the middle domain of Hsp90 and the J-protein binding site of Hsp70 in both E. coli and yeast. Moreover, we showed that DnaJ promotes the DnaK-Hsp90 interaction in the presence of ATP, likely by converting DnaK into the ADP-bound conformation, which is the conformation that binds Hsp90Ec. This work suggests that the direct interaction between Hsp90 and Hsp70 chaperones of E. coli and yeast is an intermediate in the pathway of protein remodeling and likely important in the transfer of the clients from Hsp70 to Hsp90. To gain insight into the in vivo role of Hsp90Ec in E. coli, we explored an overexpression phenotype of Hsp90Ec. We previously observed that Hsp90 causes cell filamentation when highly expressed in E. coli. We have found that cells filament when Hsp90Ec is overexpressed because FtsZ, a bacterial tubulin homolog essential for cell division, fails to assemble into FtsZ rings. In vitro, Hsp90Ec interacts with FtsZ and inhibits FtsZ polymerization. Moreover, E. coli deleted for the Hsp90Ec gene, htpG, degrade FtsZ more rapidly than wild-type cells, and the length of cells lacking htpG is reduced compared to wild-type cells. Altogether, these results suggest that Hsp90Ec is a modulator of cell division, and imply that the polypeptide-holding function of Hsp90 may be a biologically important ATP-independent chaperone activity of Hsp90Ec. Our second aim is to clarify the mechanism of action of Clp chaperones in protein remodeling and proteolysis and the regulation of Clp proteases by adaptors and anti-adaptors. Bacterial Clp proteases are comprised of an ATP-dependent chaperone component and a protease component. They have analogous structures and functions to the eukaryotic proteasome and the importance of the proteasome in cancer is well documented. E. coli ClpXP is a two-component ATP-dependent protease that unfolds and degrades proteins bearing specific recognition signals. One important substrate of ClpXP is RpoS, the stationary phase RNA polymerase sigma factor of E. coli. ClpXP, like many Clp proteases are regulated by adaptor proteins and anti-adaptor proteins. One ClpXP adaptor protein, RssB, is essential to target RpoS for degradation during exponential cell growth. In response to various stress conditions, one of the several anti-adaptor proteins, IraP, IraM or IraD, interacts with RssB to block RpoS degradation. Recent work in collaboration with Susan Gottesman's group (NCI) and Alexandra Deaconescu's group (Brown University) has demonstrated how one of the anti-adaptor proteins, IraD, physically and functionally interacts with RssB to block RpoS degradation by ClpXP. In our ongoing collaboration, we are exploring the mechanism of ClpXP regulation by RssB and the inhibition of RssB by other anti-adaptor proteins. Altogether the work is revealing the mechanisms of regulation of proteases by adaptors and anti-adaptors.